Compound and method of treatment for fungal pathologies of the oral cavity

ABSTRACT

The broadest aspect of the invention is a composition and method of treatment of fungal pathologies of the oral cavity or fungal growth on the surface of dentures. A preferred embodiment of the is a pharmacologically effective amount of a peptide selected from the group of peptides with a C-terminal sequence consisting of KPV, HFRWGKPV, and SYSMEHFRWGKPV in combination with a therapeutically effective amount of a fungicide selected from the group consisting of: itraconazole, econazole, ketoconazole, miconazole and fluconazole. Another embodiment of the invention is a method for treating fungal pathologies of the of oral cavity and dentures by application of a pharmacologically effective amount of a peptide selected from the group of peptides with a C-terminal sequence consisting of KPV, HFRWGKPV, and SYSMEHFRWGKPV in combination with a therapeutically effective amount of a fungicide selected from the group consisting of: itraconazole, econazole, ketoconazole, miconazole and fluconazole. In yet another embodiment of the invention these peptides are used in combination with a therapeutically effective amount of gram positive and/or gram negative antibiotics.

BACKGROUND OF THE INVENTION

[0001] This invention relates to the field of pathologies of the oralcavity caused by fungi.

[0002] One of the most common and stubborn infections of the mouth andthroat is Candidiasis. Thrush or acute candidiasis is caused byextensive candidal invasion of the oral mucosal epithelium. Thrushpresents as creamy, yellow tufts which can be readily wiped off with aswab to expose a red and inflamed area of epithelium. The cottony tuftsare the result of extensive infiltration of candidal hyphae into themucosal epithelium.

[0003] Although thrush is most common in infants, it also occurs inadults that are immunocompromised, undergoing broad spectrum antibiotictreatment, undergoing corticosteroid treatment, diabetics and anemics.In adult patients, particularly immunosuppresed patients, thrush istreated with large amounts of azole compounds such as miconazole,itraconazole, econazole, ketoconazole or fluconazole. Among thrushpatients not already undergoing broad-spectrum antibiotic treatment,broad-spectrum antibiotics are frequently prescribed in combination withfungicide therapy to avoid or treat any secondary bacterial infections.Infants are usually treated with fungicidal suspensions if the infectiondoes not spontaneously clear.

[0004] Chronic candidiasis is both far more common and far moredifficult to treat than thrush. Candidiasis is characterized by lessextensive innervation of the epithelium by the Candidal hyphae thanthrush.

[0005] Candidal proliferation on denture surfaces is a common, chronic,but minor form of candidiasis. Inflammation of the epithelium underdentures, denture stomatitis, may be caused by the proliferation of C.albicans in the interface between the denture-bearing mucous membraneand the denture surface. The lower denture-bearing area is typicallyfreely exposed to saliva, and consequently denture stomatitis is rarelyseen in this site. However, a close-fitting upper denture creates amicroenvironment cut-off from any protective effects of saliva. When C.albicans proliferates under a denture, it is held in prolonged contactwith the mucous membrane and presumably acts as an irritant.

[0006] Denture stomatitis is considered a common cause of inflammationof the epithelium near the corners of the mouth, angular stomatitis,among ambulatory patients. Typical treatment requires vigorous use oftopical oral nystatin or amphotericin B to resolve denture stomatitisand associated angular stomatitis. During treatment, the dentures mustbe worn as little as possible to allow the drug to the reach affectedarea, thus, inconveniencing suffering denture wearers.

[0007] Candidal leukoplakia is a less common type of chroniccandidiasis, appearing as a thick keratizined lesion on or about thetongue. Microscopically, a candidal leukoplia plaque consists of a thicklayer parakeratinized epithelium invaded by candidal hyphae. Thesekeratinized plaques of chronic candidiasis are tough, adherent, andoften irregular in thickness, persistent, and therefore, unlike the softfriable plaques of thrush. Common sites are the commissures of thetongue and cheeks. Treatment of candidal leuloplakia infections aredifficult since the intracellular growth of the candida makes it lessaccessible to antifungal drugs. Absent treatment, these leukoplakiasoften cover large areas of the mouth and tongue, making eating painfuland causing significant social anxiety among the afflicted.

[0008] A number of rare forms of chronic mucocutaneous candidiasisappear associated with various immune disorders. Familial chronicmucocutaneous candiasis is a rare, recessive, immune disorder whichconfers a tendency to develop chronic, leukoplakia-like plaques. Diffusechronic mucocutaneous candidiasis is associated with susceptibility tobacterial infections, particularly of the respiratory tract, and otherfungal infections. Its lesions may extend down the pharynx or larynx andwhen affecting the mouth and lips, can be severely disfiguring. Candidaendocrinopathy syndrome appears to be transmitted as an autosomalrecessive gene. The candidal infections tend to be mild and dermallesions are rare. The main features are the associated endocrinedeficiencies. Most common is hypoparathyroidism, but hypoadrenalism andalmost any other type of endocrine deficiency can develop.

[0009] Treatment for all these candidal immune disorders are difficult.If the immunological defect can be identified, as is sometimes the casewith diffuse chronic mucocutaneous candidiasis, treatment may bepossible, but generally treatments are ineffective because they fail toaddress the underlying immunological disorder that allows the candida toflourish.

[0010] The foregoing discussion indicates that an effective fungicideagainst a wide range of candidal pathologies useful inimmuno-compromised patients, children, and the elderly that can eitherbe topically applied or systemically applied to treat the chroniccandidal infections like candidal leukoplakia that is resistant totopical treatment would be a significant advance to the art. Such afungicidal should preferably have low toxicity and even more preferablypossess anti-inflammatory properties. Lastly, the fungicidal should alsohave minimal cross reactivity with other drugs so that it may besimultaneously prescribed with the complicated treatment regimens of theelderly and chronically ill-two groups likely to suffer candidiasis.

SUMMARY OF THE INVENTION

[0011] The invention includes a composition and method of treatment offungal pathologies of the oral cavity or fungal growth on the surface ofdentures. In a preferred embodiment of the invention a therapeuticallyeffective amount of one or more peptides selected from the group ofpeptides with a C-terminal sequence consisting of KPV, HFRWGKPV, andSYSMEHFRWGKPV used in combination with a therapeutically effectiveamount of a fungicide selected from the group consisting of:itraconazole, econazole, ketoconazole, miconazole and fluconazole anddissolved into a carrier. More preferably still each peptide has theprimary sequence of KPV or VPK-Ac-CC-Ac-KPV (Ac=Acetyl group).Pharmacologically effective concentrations may be as low as 10⁻¹² M butmay be as high 10⁻⁴ M. Pharmacologically effective concentrations ofthese peptides may be incorporated into commercial formulations ofcreams, gels, mouthwashes, toothpastes, tablets, or atomized sprays.

[0012] In another preferred embodiment of the invention these peptidesare topically or systemically applied to treat a candida infection ofthe oral cavity. In yet another embodiment of the invention thesepeptides are used in combination with a therapeutically effective amountof a gram positive or gram negative antibiotic to prevent or treatsecondary bacterial infections of the oral cavity or on the surface ofdentures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 illustrates the effect of α-MSH (1-13) and (11-13) and thepeptide VPK-Ac-CC-Ac-KPV on C. albicans colony forming units compared tocontrols. All three molecules significantly decreased C. albicans colonyforming units over a broad range of peptide concentrations.

[0014]FIG. 2 represents a comparison of candidacidal activity of certainmelanocortin peptides and fluconazole (all 10⁻⁶M). The most effective ofthe melanocortin peptides were those including the C-terminal amino acidsequence of α-MSH, namely, α-MSH (1-13), (6-13) and (11-13).

[0015]FIG. 3A shows untreated germination of C. albicans, i.e.blastopores.

[0016]FIG. 3B shows a horse serum-induced germination of C. albicans.

[0017]FIG. 3C shows the effect of α-MSH (1-13) treatment on germinationof C. albicans

[0018]FIG. 3D shows the effect of α-MSH (11-13) treatment on germinationof C. albicans

[0019]FIG. 4 illustrates the effect of α-MSH (1-13) and (11-13) on C.albicans killing by human neutrophils. Values are expressed as percentincrease in killing vs. medium along. Scores are means±SEM.

[0020]FIG. 5 illustrates the effect of α-MSH (1-13), (11-13), andforskolin on cAMP content of C. albicans.

[0021]FIG. 6 illustrates the inhibitory effect of α-MSH (1-13), (11-13),and forskolin on C. albicans colony forming units.

DETAILED DESCRIPTION OF THE INVENTION

[0022] α-MSH is a 13 amino acid, fungicidal peptide with the primarysequence SYSMEHFRWGKPV. In addition to its fungicidal properties it alsoanti-pyretic and anti-inflammatory. The C-terminal trimer, KPV, appearsresponsible for these properties. Lipton, J. M., Antipyretic andAnti-inflammatory Lys-Pro-Val- Compositions and Methods of Use, U.S.Pat. No. 5,028,592, issued Jul. 2, 1991; Lipton, J. M., Antipyretic andAnti-inflammatory Lys-Pro-Val- Compositions and Methods of Use, U.S.Pat. No. 5,157,023, issued Oct. 20, 1992; Catania, A., Lipton J. M.,α-Melanocyte Stimulating Hormone in the Modulation of Host Reactions,Endocr. Rev. 14, 564-576 (1993); Lipton, J. M., Catania, A.,Anti-inflammatory Influence of the Neuroimmunomodulator α-MSH, Immunol.Today 18, 140-145 (1997). herein incorporated by reference. Allreferences are hearby incorporated by reference in their entirety. Thecore α-MSH sequence (4-10) has learning, memory and behavioral effectsbut little anti-pyretic and anti-inflammatory activity. Lipton, J. M.,Catania, A., Anti-inflammatory Influence of the Neuroimmunomodulatorα-MSH, Immunol. Today 18, 140-145 (1997). α-MSH, the α-MSH core and itstripeptide C-terminal have very low toxicity. Lipton, J. M., Catania,A., Anti-inflammatory Influence of the Neuroimmunomodulator α-MSH.Immunol. Today 18, 140-145 (1997).

[0023] α-MSH is produced by the post translational processing ofpropriomelanocortin and shares the 1-13 primary sequence withadrenocortitrophic hormone (ACTH). Eberle, A. N., The Melanotropins,Karger, Basel, Switzerland (1988). It is secreted by a wide variety ofcell types, including pituitary cells, monocytes, melanocytes,keratinocytes, epidermal cells and the epithelial cells of mucousmembranes. Lipton, J. M., Catania, A., Anti-inflammatory Influence ofthe Neuroimmunomodulator α-MSH, Immunol. Today 18, 140-145 (1997); seealso Catania et al., unpublished.

[0024] α-MSH reduces inflammation and fever by modulating theinflammatory cascade locally and systemically. Rajora, N., Ceriani, G.,Catania, A., Star, R. A., Murphy, M. T., Lipton, J. M., α-MSHProduction, Receptors and Influence of Neopterin, in a HumanMonocyte/macrophage Cell Line, H. Leukoc. Biol. 59,248-253 (1996); Star,R. A., Rajora, N. Huang, J., Stock, R. C., Catania, A., Lipton, J. M.,Evidence of Autocrine Modulation of Macrophage Nitric Oxide Synthase byα-MSH, Proc. Natl. Acad. Sci. 92, 8016-8020 (1995); Lipton, J. M.,Ceriani, G., Macaluso, A., McCoy, D., Carnes, K., Biltz, J., Catania,A., Anti-inflammatory Effects of the Neuropepotide α-MSH in Acute,Chronic and Systemic Inflammation, Ann. N. Y. Acad. Sci. 741, 137-148(1994); Rajora, N., Boccoli, G., Burns, D., Sharma, S., Catania, A.,Lipton, J. M., α-MSH Modulates Local Circulating Tumor Necrosis Factor Ain Experimental Brain Inflammation, J., Neurosci, 17, 2181-2186 (1997);Richards, D. B., Lipton, J. M. Effect of α-MSH (11-13)(Lys-Pro-Val)onFever in Rabbits, Peptides 5, 815-817 (1984); Hiltz, M. E., Lipton, J.M., Anti-inflammatory Activity of a COOH-terminal Fragment of theNeuropeptide α-MSH, FASEB J. 3, 2282-2284 (1989).

[0025] The broadest aspect of the invention is a composition and methodof treatment of fungal pathologies of the oral cavity or fungal growthon the surface of dentures. In a preferred embodiment of the invention atherapeutically effective amount of one or more peptides selected fromthe group of peptides with a C-terminal sequence consisting of KPV,HFRWGKPV, and SYSMEHFRWGKPV is incorporated into a carrier. Morepreferably still, each peptide has the primary sequence of KPV orVPK-Ac-CC-Ac-KPV (Ac=Acetyl group). Pharmacologically effectiveconcentrations may be as low as 10⁻¹²M but may be as high 10⁻⁴ M. Apreferred embodiment of the invention utilizes peptide concentrations of10⁻¹²M to 10⁻¹⁰ M. Pharmacologically effective concentrations of thesepeptides may be incorporated into commercial formulations of creams,gels, mouthwashes, toothpastes, tablets, or atomized sprays.

[0026] Formulations of creams and gels are well known in the art.Harry's Comseticology (Chemical Publishing, 7^(th) ed. 1982);Remington's Pharmaceutical Sciences (Mack Publishing Co., 18^(th) ed.1990).

[0027] Set forth below are examples of various formulations of theinvention. As used below the term “Active ingredient” refers to one ormore peptides selected from the group of peptides with a C-terminalsequence consisting of KPV, HRFWGKPV and SYSMEHFRWGKPV. Preferably, theactive ingredient is KPV or VPK-Ac-CC-Ac-KPV.

[0028] An exemplary formulation of a gel based on the inventioncomprises: Propylene Glycol 10.0 g PEG-Glyceryl Cocoate 10.0 gdi-α-Tocopherol  .02 g Ascorbyl Palmitate  .10 g Propyl Gallate .002 gCitric Acid, annhydr  .01 g Isopropanol 50.0 g Hydroxypropyl Methyl 3.00g Cellulose Water 100 g Active ingredient .2-.2*10⁻⁸ mg

[0029] An exemplary formulation of a cream comprises: Glycerol  5.0 gNa₂-EDTA .03 g Glycerides 10.0 g  Cetyl Alcohol 1.0 g Stearyl Alcohol1.0 g Glycerol mono Stearate 4.0 g Cetereth 2.0 g di-α-tocopherol .02 gWater 100.0 g  Active Ingredient .1-.1*10⁻⁸ mg

[0030] Formulations for toothpastes, and mouthwashes are well known inthe art. U.S. Pat. Nos. 4,719,100; 4,314,990 and 4, 151,271. Anexemplary formulation of a toothpaste comprises: Sorbitol (70% aq.) 52.0g  Sodium Saccharine  .3 g Trisodium Phosphate 1.1 g Precipitated Silica20.0 g  Glycerine 18.0 g  Sodium Flouride  .3 g Water 3.0 g Sodium AlkylSulfate (28.8 aq.) 4.0 g Flavoring 1.0 g Active Ingredient .2-.2*10⁻⁸ mg

[0031] An exemplary formulation of a mouthwash comprises: Water 89.0 g 1-methoxypropanol 7.0 g n-propanol 1.0 g Saccharine .06 g Glycerol 1.3 gFlavoring 1.0 g VPK-Ac-CC-Ac-KPV dimer .1-.1*10⁻⁸ mg

[0032] In another preferred embodiment of the invention thesecompositions are topically or systemically applied to treat a candidainfection of the oral cavity. Topical administration may be made withmanual application of creams or gels, gargling of mouthwash, brushingwith toothpaste, chewing on tablets, holding lozenges in the mouth orwith an atomized spray. Systemic administration may be made by ingestionof hard tablets, soft tablets or capsules. In yet another preferredembodiment of the invention these peptides are used in combination witha therapeutically effective amount of a fungicide selected from thegroup consisting of: itraconazole, econazole, ketoconazole, miconazoleand fluconazole. In yet another preferred embodiment of the inventionthese peptides are used in combination with a therapeutically effectiveamount of a gram positive or gram negative antibiotics selected from thegroup consisting of: aminglycosides, amoxicillin, ampicillin,azithramycin, erythromycin, nafcillin, penecillin, quinupuristindalfopristin and vancomycin.

[0033] The formulation of tablets are well known in the art. Anexemplary formulation of a hard gelatinous tablet comprises: GelatineBloom 30 70.0 mg Maltodextrin MD 05 108.0 mg di-α-tocopherol 2.0 mgSodium ascorbate 10.0 mg Microcrystalline cellulose 48.0 mg Magneisumstearate 2.0 mg Active Ingredient .2*10⁻⁴-.2*10⁻¹¹ mg

[0034] An exemplary formulation of a hard tablet comprises: Annhydrouslactose 130.5 mg  Microcrystalline cellulose 80.0 mg  di-α-tocopherol2.0 mg Sodium ascorbate 10.0 mg  Polyvinylpyrrolidone K30 5.0 mgMagnesium stearate 2.0 mg Active Ingredient .2*10⁻⁴-.2*10⁻¹¹ mg

[0035] The following examples teach the utility of a-MSH as a fungicidein general and anti-candidal fungicide in particular. Methods inmicrobiology, molecular biology and biochemistry used but not explicitlydescribed in this disclosure are amply described throughout theliterature and well within the ability of one skilled in the art.

[0036] The peptides used in the following examples include: α-MSH (1-13)(SEQ. ID. NO. 4), (4-10) (SEQ. ID. 002), (6-13) (SEQ. ID. 003), and(11-13) (SEQ. ID. NO. 1), all of which N-acetylated and C-amidated, andACTH (1-39) and (18-39) (CLIP). These peptides were prepared bysolid-phase peptide synthesis and purified by reversed phased highperformance liquid chromatography. Some examples also include a dimer ofthe amino acid sequence KPV (SEQ. ID. NO. 1), VPK-Ac-CC-Ac-KPV (SEQ. ID.NO. 8), which also was N-acetylated and C-amidated (KPV dimer). Dimerscan be formed by adding cysteines at the N-termini of any of the abovepolypeptides and allowing the cysteines of two polypeptides to form adisulfide bond. Both homo-dimers and hetero-dimers can be formed usingthis method.

[0037]C. albicans (clinical isolate) was obtained from the collection ofthe Department of Microbiology, Ospedale Maggiore di Milano andmaintained on Sabouraud's agar slants and periodically transferred toSabouraud's agar plates and incubated for 48 hours at 28° C. To preparea stationary growth phase yeast, a colony was taken from the agar plateand transferred into 30 ml Sabouraud-dextrose broth and incubated for 72hours at 32° C. Cells were centrifuged and suspended in Hank's balancedsalt solution (“HBSS”) to the desired concentration. Viability,determined by the exclusion of 0.01% methylene blue, remained >98%.

[0038] Statistical significance disclosed in the examples below wasanalyzed using one-way analysis of variance and the Student's t test.Probability values greater than 0.05 were considered significant.

EXAMPLE 1

[0039] The first example suggests that α-MSH (11-13), (6-13) and (1-13)exhibit similar anti-candidal properties as flucanazole over anexceedingly broad range of concentrations.

[0040]C. albicans (1×10⁶/ml in HBSS) was incubated in the presence ofabsence or α-MSH (1-13) or (11-13) at concentrations in the range of10⁻¹⁵ M to 10⁻⁶ M for 2 hours at 37° C. Cells were then washed in colddistilled waste and diluted with HBSS to a concentration of 100organisms/ml. One-ml aliquots were dispensed on blood agar plates andincubated for 48 hours at 37° C. Organism viability was estimated fromthe number of colonies formed.

[0041] In subsequent experiments using familiar procedures we comparedactivity of α-MSH (4-10), (6-13), (11-13), ACTH (1-39), (18-39) andfluconazole, the latter an established antifungal agent. Melanocortinpeptides and fluconazole were tested in concentrations of 10⁻⁶ M to 10⁴M. There were at least six replicates for each concentration of peptide.

[0042]FIG. 1 shows that C. albicans colony forming units (CFU) weregreatly reduced by α-MSH (1-13) and (11-13). FIG. 1 also shows that theVPK-Ac-CC-Ac-KPV peptide also inhibited C. albicans colony formation).Concentrations of all three peptides from 10⁻¹²M to 10⁻⁴M hadsignificant inhibitory effects on CFU (p<0.01 vs. control).

[0043]FIG. 2 demonstrates that in experiments comparing the relativepotency of 10⁻⁴M melanocortin peptides in reducing C. albicansviability, α-MSH (11-13), (6-13) and (1-13) were the most effective.Their inhibitory activity was similar to that of equimolar fluconazole.The core α-MSH sequence (4-10), which has behavioral effects but littleanti-inflammatory activity, caused approximately 50% inhibition of CFU.FIG. 2 also shows that although this inhibitory effect was substantial(p<0.01 vs. control), it was significantly less than that caused byα-MSH fragments bearing the KPV signal sequence, i.e., α-MSH (6-13) and(11-13) (p<0.01), or the parent molecule α-MSH (1-13)(p<0.05). ACTH(1-39) and the ACTH fragment (18-39) did not reduce C. albicansviability. Even higher concentrations of these ACTH peptides (up to10⁻⁴M) were likewise ineffective in reducing C. albicans CFU (resultsnot shown in the figures).

[0044] These results show that α-MSH(1-13), its C-terminal tripeptide(11-13), and other α-MSH fragments have significant fungicidal effectsagainst C. albicans. The most effective of the α-MSH peptides were thoseincluding the C-terminal amino acid sequence KPV of the α-MHS sequence,i.e., α-MSH (1-13), (6-13) and (11-13). In addition, the sequenceVPK-Ac-CC-Ac-KPV has also been shown to be at least as effective α-MSH(11-13) against microbes. The α-MSH cores sequence (4-10), which isknown to influence learning and memory, but has little antipyretic andanti-inflammatory influence, was effective, but less so. The ACTHpeptides (1-39) and (18-39) did not have significant candidacidaleffects. These observations indicate that antifungal activity is notcommon to all melanocortin peptides, but rather that is specific toα-MSH amino acid sequences, and most particularly to the C-terminalamino-acid sequences of α-MSH. This strongly suggests that α-MSH(1-13),its C-terminal tripeptide (11-13), and other α-MSH fragments couldserver as a basis for a therapeutic treatment of acute and chroniccandidal infections of the oral cavity or as antifungal agent againstcandidal growth on denture surfaces.

EXAMPLE 2

[0045] Example 2 demonstrates that α-MSH (1-13), (6-13) or (11-13)strongly inhibits Candidal germination. C. albicans from stationaryphase cultures were washed twice with distilled water and suspended inHBSS to a final concentration of 2×10⁶/ml. Hyphal growth was induced byaddition of 10% inactivated horse serum (GIBCO/BRL, Great Britain) toyeast incubated for 45 minutes at 37° C. with continuous shaking. Horseserum was removed by washing cells twice with HBSS and incubation wascontinued for 60 minutes at 37° C. in the presence of α-MSH (1-13),(6-13) or (11-13) at a concentration of 10⁻⁶M with continuous shaking.The percentage of filamentous cells was evaluated under a lightmicroscope with the aid of hemocytometer. Experiments were run intriplicate and at least 200 cells were scored. Photomicrographs weretaken with a MC100 camera attached to an Axioskop Zeiss microscope.

[0046] FIGS. 3A-D show that coincubation of C. albicans with α-MSH(1-13) or (11-13) inhibited germ tube formation induced by horse serum,α-MSH (1-13) caused 28-32% reduction in the number of filamentous cells;the tripeptide inhibited germination by 54-58%. The octapeptide α-MSH(6-13) had similar activity (approximately 50% inhibition)(not shown).

[0047] The pathogenesis of C. albicans infection involves adhesion ofyeast cells to epithelial cells, commonly found in the mucosal membranesof the ears, eyes, nose and throat and/or endothelial cells, followed bymorphologic switching of the yeast cells from the ellipsoid blastosporeto various filamentous forms: germ tubes, pseudohyphae and hyphae. Gow,N. A., Germ Tube Growth of Candida Albicans, Curr. Topics Med. Mycol. 8,43-45 (1997). The results also show that in addition to directcandicidal properties, α-MSH(1-13), its C-terminal tripeptide (11-13),and other α-MSH fragments interfere with germination and adhesion ofcandida to the epithelium. This suggests that if the germination andadhesion of candida could be interfered with, the invasive forms ofchronic candidiasis that innervate the epithelium could be treated withtherapy based upon α-MSH(1-13), its C-terminal tripeptide (11-13), andother α-MSH fragments.

EXAMPLE 3

[0048] Example 3 illustrates that α-MSH and its derivatives exhibittheir anti-candidal properties without compromising the ability of humanneutrophils to independently combat Candida. Venous blood (20 ml) fromhealth volunteers was anticoagulated with heparin. Neutrophils wereisolated using dextran sedimentation and Ficoll-Hypaque (Sigma ChemicalCo., St. Louis, Mich., USA) centrifugation. Erythrocytes were lysed viahypotonic shock. Neutrophils represented at least 97% of the cellsuspension. Cell viability, estimated by trypan blue exclusion,was >98%. Neutrophils were suspended to a final concentration in HBSS.

[0049]C. albicans (1×10⁶) were opsonized with human AB serum in ashaking water bath for 30 minutes at 37° C. Organisms were thenincubated with neutrophils in medium or in medium with α-MSH (1-13) orα-MSH (11-13) in concentrations of 10⁻¹⁵ M to 10⁻⁴ M in a shaking waterbath for 2 hours at 37° C. After incubation, the culture tubes wereplaced on ice to stop growth and extracellular organisms were washedtwice with centrifugation at 1000×g at 4° C. A 2.5% sodiumdesoxycholoate solution was added to obtain a suspension of 10⁶cells/ml. Two 1/100 serial dilutions in HBSS were made to obtain a finalsuspension of 100 cells/ml. Aliquots of 1 ml were dispensed on bloodagar plates and incubated for 48 hours at 37° C. Colony forming units(CFUs) were counted at the end of the incubation period. Experimentswere run in triplicate and repeated using blood from 5 different donors.

[0050]FIG. 4 shows that α-MSH(1-13) and (11-13) enhanced the killing ofC. albicans by human neutrophils when administered in concentrations of10⁻¹²M to 10⁻⁴M (p<0.01). Therefore, enhanced killing occurred over avery broad range of concentrations including picomolar concentrations,i.e. the quantity of α-MSH found in human placenta. Catania, A.,Airaghi, L., Garofalo, L., Cutuli, M., Lipton, J. M., The Neuropeptide αMSH in AIDS and Other Conditions in Humans, Ann. N.Y. Acad. Sci. 840,848-856 (1998).

[0051] Reduced killing of pathogens is a dire consequence of therapywith corticosteroids and nonsteroidal anti-inflammatory drugs duringinfection. Stevens, D. L., Could Nonsteroidal Anti-inflammatory Drugs(NSAIDS) Enhance Progression of Bacterial Infections to Toxic ShockSyndrome?, Clin. Infect. Dis., 21, 977-80 (1997); Capsoni, F., Meroni,P. L., Zocchi, M. R., Plebani, A. M., Vezio, M., Effect ofCorticosteroids on Neutrophil Function: Inhibition of Antibody-dependentCell-mediated Cytotoxicity (ADCC), J. Immunolpharmacol. 5, 217-230(1983). This effect is particularly dangerous in immunocompromisedpatients.

[0052] These results also suggest that α-MSH(1-13), its C-terminaltripeptide (11-13), and other α-MSH fragments would be useful fortreatment of candidiasis in immunocompromised patients since thesepeptides appear not to reduce neutrophil chemotaxis and thus would notfurther comprise the immune system.

EXAMPLE 4

[0053] Example 4 suggests a cellular mechanism to explain how α-MSHexerts its anti-candidal properties. C. albicans (10⁶/ml), permeabilizedwith toluene/ethanol, were incubated at 37° C. with continuous shakingin the presence of 10⁻⁶ M α-MSH (1-13), (11-13), forskolin, an agentknown to increase intracellular cAMP, or in medium alone. The reactionwas stopped after 3 minutes by the addition of ice cold ethanol, cAMPwas measured in duplicate using a commercial enzyme immunoassay (EIA)kit (Amersham, United Kingdom) after extraction via the liquid-phasemethod according to manufacturer's instructions. The effect of forskolin(10⁻⁶ M) on C. albicans colony formation was determined using the sameprocedure as for α-MSH peptides.

[0054] Because many of the effects of α-MSH are known to be mediated byinduction of cAMP, we measured effects of α-MSH peptides on cAMPaccumulation in C. albicans . FIG. 5 shows that α-MSH (1-13) and (11-13)enhanced cAMP content in the yeast. FIG. 6 shows the increase was of thesame order of magnitude as that induced by equimolar forskolin, anadenylate cyclase activator. To determine whether increases in cAMPcould be responsible for reduction in CFU, we tested the effects offorskolin on C. albicans viability. Results showed that 10⁻⁶M forskolinmarkedly inhibited C. albicans CFU relative to control (p<0.01). FIG. 6demonstrates that the inhibitory effect was similar to that exerted byα-MSH.

[0055] The mechanism of action of natural antimicrobial agents is onlypartly understood. Most of these peptides, including the defensins,alter membrane permeability and impair internal homeostasis of theorganism. The first contact is made between the cationic groups of thepeptide and the negatively charged head of the target membrane. Then,the tertiary structure determines the mode of insertion of the peptideinto membranes where they form ion channels or pores that disrupt cellintegrity. It is known that cAMP-enhancing agents inhibit MRNA andprotein synthesis in C. albicans . Bhattacharya, A., Datta, A., Effectof Cyclic AMP on RNA and Protein Synthesis in C. albicans, Biochem.Biophys. Res. Commun. 77: 1483-44 (1977).

[0056] In the present experiments it is shown that α-MSH induces cAMPaccumulation in C. albicans and also that the cAMP-inducing agentforskolin inhibited colony formation. Without being limited by thistheoretical explanation, it may be that the antimicrobial effect wascaused by enhancement of this mediator.

EXAMPLE 5

[0057] Example 5 suggests functional equivalents to α-MSH and itsderivatives. As used herein, a biological functional equivalent isdefined as an amino acid sequence that is functionally equivalent interms of biological activity.

[0058] Although the specific amino acid sequences described here areeffective, it is clear to those familiar with the art that amino acidscan be substituted in the amino acid sequence or deleted withoutaltering the effectiveness of the peptides. Further, it is known thatstabilization of a the α-MSH sequence can greatly increase the activityof the peptide and that substitution of amino acid D-forms for L-formscan improve or decrease the effectiveness of peptides. For example, astable analog of α-MSH, [Nle⁴,D-Phe⁷]-α-MSH which known to have markedbiological activity on melanocytes and melanoma cells, is approximately10 times more potent than the parent peptide in reducing fever.Holdeman, M., and Lipton, J. M., Antipyretic Activity of a Potent α-MSHAnalog, Peptides 6, 273-5 (1985). Further, adding amino acids to theC-terminal α-MSH (11-13) sequence can reduce or enhance antipyreticpotency. Deeter, L. B., Martin, L. W., Lipton, J. M., AntipyreticProperties of Centrally Administered α-MSH Fragments in the Rabbit,Peptides 9, 1285-8 (1989). Addition of glycine to form the 10-13sequence slightly decreased the potency; the 9-13 sequence was almostdevoid of activity, whereas the potency of the 8-13 sequence was greaterthan that of the 11-13 sequence. It is known that Ac-[D-K¹¹]-α-MSH11-13-NH₂ has the same general potency as the L-form of the tripeptideα-MSH 11-13. Hiltz, M. E., Catania, A., Lipton, J. M., Anti-inflammatoryActivity of α-MSH (11-13) Analogs Influences of Alterations inStereochemistry, Peptides 12, 767-71 (1991). However, substitution withD-proline in position 12 of the tripeptide rendered it inactive.Substitution with the D-form of valine position 13 or with the D-form oflysine at position 11 plus the D-form of valine at position 13 resultedin greater anti-inflammatory activity than with the L-form tripeptide.These examples indicate that alterations in the amino acidcharacteristics of the peptides can influence activity of the peptidesor have little effect, depending upon the nature of the manipulation.

[0059] It is also believed that biological functional equivalents may beobtained by substitution of amino acids having similar hydropathicvalues. Thus, for example, isoleucine and leucine, which have ahydropathic index +4.5 and +3.8 , respectively, can be substituted forvaline, which has a hydropathic index of +4.2, and still obtain aprotein having like biological activity. Alternatively, at the other endof the scale, lysine (−3.9) can be substituted for arginine (−4.5), andso on. In general, it is believed that amino acids can be successfullysubstituted where such amino acid has a hydropathic score of withinabout +/−1 hydropathic index units of the replaced amino acid.

EXAMPLE 6

[0060] An elderly diabetic patient presents with white plaque likelesions on the tongue. The patient has been a diabetic since childhoodwith a poor history of blood sugar control. Samples of the whitekeratinized lesions show moderate candidal fungal hyphae innervation.The patient is prescribed a pharmacologically effective amount ofamphotericin and a pharmacologically effective concentration of α-MSH.

[0061] Examples 1-6 demonstrate the anti-candidal properties and uses ofα-MSH and/or its derivatives. These are only illustrative and are notintended to limit the invention. It is understood that modifying theexamples above does not depart from the spirit of the invention. It isfurther understood that the examples can be applied independently or incombination with each other.

What is claimed is:
 1. A pharmaceutical composition for the treatment offungal pathologies of the oral cavity comprising a therapeuticallyeffective amount of a peptide having a C-terminal sequence amino acidsequence KPV (SEQ. ID. NO. 1) in combination with a therapeuticallyeffective amount of a fungicide.
 2. The pharmaceutical composition ofclaim 1 wherein the peptide is selected from the group of peptides witha C-terminal amino acid sequence consisting of: KPV (SEQ. ID. NO. 1),VPK-Ac-CC-Ac-KPV (SEQ. ID. NO. 8), HFRWGKPV (SEQ. ID. NO. 3) andSYSMEHFRWGKPV (SEQ. ID. NO. 4).
 3. The composition of claim 1 whereinsaid fungicide is selected from the group consisting of: itraconazole,econazole, ketoconazole, miconazole and fluconazole.
 4. The compositionof claim 1 further comprising a therapeutically effective amount of anantibiotic.
 5. The composition of claim 4 wherein said antibioticfurther comprises a gram negative antibiotic.
 6. The composition ofclaim 4 wherein said antibiotic further comprises a gram positiveantibiotic.
 7. The composition of claim 4 wherein said antibiotic isselected from the group consisting of: aminglycosides, amoxicillin,ampicillin, azithramycin, erythromycin, nafcillin, penecillin,quinupuristin dalfopristin and vancomycin.
 8. The pharmaceuticalcomposition of claim 1 further solvated in a carrier.
 9. The compositionof claim 1 wherein the concentration of said peptide is at least 10¹²M.10. The composition of claim 8 wherein the carrier is selected from thegroup consisting of: creams, gels, mouthwashes, toothpastes, tablets,and atomized sprays.
 11. A pharmaceutical composition for the treatmentof fungal pathologies of the oral cavity comprising a therapeuticallyeffective amount of a peptide having a C-terminal sequence amino acidsequence KPV (SEQ. ID. NO. 1) in combination with a therapeuticallyeffective amount of a fungicide and a therapeutically effective amountof an antibiotic.
 12. The pharmaceutical composition of claim 11 whereinthe peptide is selected from the group of peptides with a C-terminalamino acid sequence consisting of: KPV (SEQ. ID. NO. 1),VPK-Ac-CC-Ac-KPV (SEQ. ID. NO. 8), HFRWGKPV (SEQ. ID. NO. 3) andSYSMEHFRWGKPV (SEQ. ID. NO. 4).
 13. The composition of claim 11 whereinsaid fungicide is selected from the group consisting of: itraconazole,econazole, ketoconazole, miconazole and fluconazole.
 14. The compositionof claim 11 wherein said antibiotic further comprises a gram positiveantibiotic.
 15. The composition of claim 11 wherein said antibioticfurther comprises a gram negative antibiotic.
 16. The composition ofclaim 11 wherein said antibiotic is selected from the group consistingof: aminglycosides, amoxicillin, ampicillin, azithramycin, erythromycin,nafcillin, penecillin, quinupuristin dalfopristin and vancomycin. 17.The pharmaceutical composition of claim 11 further solvated in acarrier.
 18. The composition of claim 11 wherein the concentration ofsaid peptide is at least 10⁻¹²M.
 19. The composition of claim 17 whereinthe carrier is selected from the group consisting of: creams, gels,mouthwashes, toothpastes, tablets, and atomized sprays.
 20. A method forthe treatment of fungal pathologies of the oral cavity comprisingapplication of a pharmaceutical effective amount of a peptide having aC-terminal sequence amino acid sequence KPV (SEQ. ID. NO. 1).
 21. Themethod of claim 20 wherein the peptide is selected from the group with aC-terminal amino acid sequence consisting of KPV (SEQ. ID. NO. 1),VPK-Ac-CC-Ac-KPV (SEQ. ID. NO. 8), HFRWGKPV (SEQ. ID. NO. 3) andSYSMEHFRWGKPV (SEQ. ID. NO. 4).
 22. The method of claim 20 furthercomprising application of therapeutically effective amount of afungicide.
 23. The method of claim 22 wherein said fungicide is selectedconsisting of: itraconazole, econazole, ketoconazole, miconazole andfluconazole.
 24. The method of claim 20 further comprising applicationof a therapeutically effective amount of an antibiotic.
 25. The methodof claim 24 wherein said antibiotic further comprises a gram positiveantibiotic.
 26. The method of claim 24 wherein said antibiotic furthercomprises a gram negative antibiotic.
 27. The method of claim 24 whereinthe gram positive and gram negative antibiotics are selected from thegroup consisting of: aminglycosides, amoxicillin, ampicillin,azithramycin, erythromycin, nafcillin, penecillin, quinupuristindalfopristin and vancomycin.
 28. The method of claim 20 wherein saidfungal pathology is candidiasis.